pNK Cells Research Articles

Cytolytic capacity of KIR2DS1+ decidual natural killer cells in the fetal-maternal interface (P3203)

Abstract Decidual natural killer cells (dNK) are the most abundant lymphocytes at the fetal-maternal interface. dNK have been shown to be less cytotoxic but to secrete more cytokines and growth factors that may help trophoblast invasion than peripheral blood NK cells (pNK). Previous studies have shown that mothers expressing the activating Killer Immunoglobulin-like Receptor-S1 (KIR2DS1) are protected from pregnancy complications when carrying a fetus expressing more HLA-C2 group alleles than them (Hiby et al, 2010). However, no experimental studies have addressed the mechanisms involved in this beneficial effect. In this study, we demonstrate that a significantly higher percentage of dNK express KIR2DS1 and KIR2DL1 than pNK cells. dNK and pNK express similar levels of perforin and granzyme B but dNK contain increased granulysin levels. Although pNK are more cytotoxic than dNK against HLA negative targets, the total pNK cytotoxicity is efficiently inhibited by HLA-C1+ and HLA-C2+ targets. In contrast, HLA-C2+ targets only inhibit the cytotoxicity of total dNK from KIR2DS1- but not KIR2DS1+ mothers. Assessment of single KIR2DL1+ and KIR2DS1+ pNK and dNK shows that HLA-C2 efficiently inhibits KIR2DL1+ but not KIR2DS1+ cell cytotoxicity. These results suggest a dominant role for KIR2DS1+ cells in the total dNK population. Future studies will focus on the influence of the fetal/maternal HLA-C genotype on KIR2DS1+ cell function and the identification of cytokines secreted by KIR2DS1+ dNK.

Characterization and ex vivo Expansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3− placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3−, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46, and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile, immunophenotype, and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.

Activation of NK cell cytotoxicity by the natural compound 2,3-butanediol

The natural compound 2,3-BTD has diverse physiological effects in a range of organisms, including acting as a detoxifying product of liver alcohol metabolism in humans and ameliorating endotoxin-induced acute lung injury in rats. In this study, we reveal that 2,3-BTD enhances NK cell cytotoxic activity in human pNK cells and NK92 cells. Treatment of NK cells with 2,3-BTD increased perforin expression in a dose-dependent manner. This was accompanied by elevated JNK and ERK1/2 MAPK activities and enhanced expression of NKG2D/NCRs, upstream signaling molecules of the MAPK pathways. The 2,3-BTD effect was inhibited by pretreatment with inhibitors of JNK (SP) or ERK1/2 (PD) or by depleting NKG2D/NCRs or JNK1 or ERK2 with siRNA. These results indicate that 2,3-BTD activates NK cell cytotoxicity by NKG2D/NCR pathways and represent the first report of the 2,3-BTD effect on activation of innate immunity cells.

Genetic regulation on ex vivo differentiated natural killer cells from human umbilical cord blood CD34+ cells

Natural killer (NK)-cells are a lymphocyte population playing a critical role in the immune surveillance against tumors and virally infected cells. The development of human hematopoietic stem cells (hHSC) into fully differentiated NK-cells pass through discrete stages of differentiation involving a variety of factors such as cytokines, membrane factors, and transcription factors (TFs). Because there is lack of studies in this field, we decided to perform an extended analysis of TFs during in vitro differentiation of NK-cells. At several points of differentiation, cells were characterized by their mRNA expression either for NK-cell cell markers, for a number of mature NK-cell receptors or a large panel of TFs. Our data suggests that some TFs (ID2, EGR-2 and T-BET) play a role in NK-cell commitment, differentiation and maturation. Although delayed on its expression, BLIMP1 also seems to be involved in differentiation and maturation of NK cells, but not in NK-cell commitment. E4BP4 and TOX are more related with initial stages of NK-cell commitment. PU.1, MEF, Ikaros, EGR-1, BCL11B and IRF-2 revealed less involvement in maturation and were more associated with NK-cell commitment and pNK cell production. GATA-3 showed a differential role during the ontogeny of NK-cells. We show that assessment of the transcripts present in the differentiating NK-cells demonstrated, a pattern of preserved and differential gene expression remarkably similar to that seen in mice except for E4BP4 that showed constant downregulation throughout the culture period. A thorough understanding of NK-cell developmental mechanisms is important as it may enable future therapeutic manipulation.

The effect of pregnancy on the uterine NK cell KIR repertoire

The major leukocyte population in the decidua during the first trimester of pregnancy consists of NK cells that express receptors capable of recognizing MHC class I molecules expressed by placental trophoblast. These include members of the killer immunoglobulin-like receptor (KIR) family, the two-domain KIR (KIR2D), which recognize HLA-C. Interactions between decidual NK (dNK) cell KIR2D and placental HLA-C contribute to the success of pregnancy and dNK cells express KIR2D at higher frequency than peripheral NK (pNK) cells. Thus, they are biased toward recognizing HLA-C. In order to investigate when this unusual KIR repertoire appears, we compared the phenotype of NK cells isolated from non-pregnant (endometrium) and pregnant (decidua) human uterine mucosa. Endometrial NK (eNK) cells did not express KIR2D at a higher level than matched pNK cells, so the bias toward HLA-C recognition occurs as a response to pregnancy. Furthermore, HLA-C expression was upregulated on uterine stromal cells as the mucosa transformed from endometrium to decidua at the onset of pregnancy. As uterine NK (uNK) cells can mature from NK precursors and acquire KIR expression in utero, the pregnancy-specific bias of uNK cells toward HLA-C recognition could arise as developing uNK cells interact with uterine stromal cells, which express higher levels of HLA-C during pregnancy.

Natural killer cells and pregnancy outcomes in women with recurrent miscarriage and infertility: a systematic review

Peripheral natural killer (pNK) and uterine NK (uNK) cells have been associated with reproductive failure. We systematically reviewed the literature to assess whether numbers or activity of pNK or uNK cells predicted subsequent pregnancy and outcome. We searched the electronic MEDLINE database from 1950 to April 2010 for relevant publications by using MeSH terms 'natural killer cells', 'reproduction' and 'pregnancy complications'. We included studies that measured pre-pregnancy pNK and uNK cell numbers or activity in women with recurrent miscarriage (RM) or infertility, and reported subsequent pregnancy outcomes of miscarriage or failure to conceive after assisted reproductive technology (ART). The search identified 783 publications and 12 fulfilled the inclusion criteria. There were too few women entered into the observational studies to assess whether high pNK cell percentages or activity predicted subsequent miscarriage in women with idiopathic RM (numbers: n = 32, OR 17, 95% CI 0.82-350.6, activity: n = 92, OR 2.51, 95% CI 0.16-40.29), or implantation failure (n = 203, OR 1.35, 95% CI 0.28-6.46), or miscarriage in infertile women after ART (n = 79, OR 2.48, 95% CI 0.50-12.32). Similarly, the studies of uNK cells were not large enough to assess whether abnormal uNK cell density predicted subsequent miscarriage in women with idiopathic RM (n = 72, OR 1.33, 95% CI 0.16-11.11). None of the uNK cell studies in women with infertility reported pregnancy outcomes dichotomized for uNK cell numbers. The prognostic value of measuring pNK or uNK cell parameters remains uncertain. More studies are needed to confirm or refute the role of NK cell assessments as a predictive test for screening women who may benefit from immunotherapy.

Crosstalk between decidual NK and CD14+ myelomonocytic cells results in induction of Tregs and immunosuppression.

Regulatory T cells (Tregs) are thought to play a major role in pregnancy by inhibiting the maternal immune system and preventing fetal rejection. In decidual tissues, NK cells (dNK) reside in close contact with particular myelomonocytic CD14(+) (dCD14(+)) cells. Here we show that the interaction between dNK and dCD14(+) cells results in induction of Tregs. The interaction is mediated by soluble factors as shown by transwell experiments, and the prominent role of IFN-gamma is revealed by the effect of a neutralizing monoclonal antibody. Following interaction with dNK cells, dCD14(+) cells express indoleamine 2,3-dioxygenase (IDO), which, in turn, induces Tregs. Notably, unlike peripheral blood NK (pNK) cells, dNK cells are resistant to inhibition by the IDO metabolite L-kynurenine. "Conditioned" dCD14(+) cells also may induce Tregs through transforming growth factor-beta (TGF-beta) production or CTLA-4-mediated interactions, as indicated by the effect of specific neutralizing Abs. Remarkably, only the interaction between dNK and dCD14(+) cells results in Treg induction, whereas other coculture combinations involving either NK or CD14(+) cells isolated from peripheral blood are ineffective. Our study provides interesting clues to understanding how the crosstalk between decidual NK and CD14(+) cells may initiate a process that leads to Treg induction and immunosuppression. Along this line, it is conceivable that an impaired function of these cells may result in pregnancy failure.

ORIGINAL ARTICLE: Peripheral Blood NK Cells Reflect Changes in Decidual NK Cells in Women With Recurrent Miscarriages

Citation Park DW, Lee HJ, Park CW, Hong SR, Kwak‐Kim J, Yang KM. Peripheral blood NK cells reflect changes in decidual NK cells in women with recurrent miscarriages. Am J Reprod Immunol 2010; 63: 173–180Problem We aimed to investigate if peripheral blood natural killer (pNK) cell levels are correlated with decidual NK (dNK) cell levels, and if chemokine expression has any role in dNK cell regulation.Method of study Decidual tissues of women having two or more miscarriages with normal karyotype were collected after miscarriage and an immuno‐histochemisty study was made. pNK cells were evaluated using flow cytometric analysis.Results The %CD3−/56+ and %CD3−/56+/16+ pNK cells showed a significant correlation with mean number of CD56+ dNK cells. The number of decidual CD16+ cells was significantly higher in women with elevated pNK (≥15%) than that of normal pNK (&lt;15%). The %CD3−/56+ and %CD3−/56+/16+ pNK cells showed an inverse correlation with duration of gestation. The CCL3+ and CXCL12+ cells were present in the decidua; however, staining intensity was not correlated with number of dNK cells.Conclusion The pNK cell levels reflect changes in dNK cell levels. This implicates that pNK cell level is a clinically useful marker to predict pregnancy outcome. Further study is needed to examine if elevated pNK cells enhance recruitment of dNK cells in the decidua.

The genotype of the NK cell receptor, KIR2DL4, influences INF secretion by decidual natural killer cells

Natural killer (NK) cells are the predominant leukocyte in first trimester decidua and play a role in vascular remodelling through interferon gamma (IFNgamma) secretion. Membrane expression of the killer immunoglobulin-like receptor (KIR) KIR2DL4 on peripheral blood NK (pNK) cells is controlled by the 9A/10A transmembrane genetic polymorphism. On peripheral NK cells (pNK), KIR2DL4 can only be detected on the membrane of cells from individuals with at least one copy of the 10A allele and ligation of KIR2DL4 results in IFNgamma secretion. In this study, we assessed KIR2DL4 expression and IFNgamma secretion as a result of KIR2DL4 ligation, by decidual NK (dNK) cells. The 9A/10A transmembrane polymorphism was shown to control KIR2DL4 expression by dNK, as previously shown for pNK cells. Freshly isolated dNK cells from subjects with at least one 10A allele expressed KIR2DL4 whereas those from 9A homozygous subjects did not. Although freshly isolated dNK did not secrete IFNgamma in response to KIR2DL4 ligation regardless of KIR2DL4 genotype, activation by in vitro culture with IL-2 enabled dNK cells from individuals with at least one 10A allele, but not those without a 10A allele, to secrete IFNgamma in response to KIR2DL4 ligation. This study confirms that expression of KIR2DL4 by dNK is dependent on the 9A/10A polymorphism and that this polymorphism influences IFNgamma secretion by dNK cells.

Characterization of decidual leukocyte populations in cynomolgus and vervet monkeys

The objective of this study was the phenotypic and functional evaluation of decidual immune cells in the cynomolgus and vervet monkeys. Early pregnancy (days 36–42) deciduas were obtained by fetectomy for histological evaluation and decidual mononuclear leukocyte (MNL) isolation. While peripheral NK (pNK) cells in these species do not express CD56, CD56 + NK cells were abundant in decidual samples. The majority of decidual NK (dNK) cells (>80%) had high light-scatter characteristics and were CD56 brightCD16 + cells with no or very low levels of natural cytotoxicity receptors (NKp46, NKp30) and NKG2A, while a minor population were small CD56 dimCD16 − lymphocytes also expressing less NKp46, NKp30 and NKG2A than pNK cells. All dNK cells were found to be perforin +; however, their cytotoxic potential was low and cynomolgus dNK cells showed strongly reduced cytotoxicity against target cells compared with pNK cells. Macrophages and T cells together comprised approximately 25–30% of decidual MNL. Decidual T cells contained a higher proportion of the minor T cell subtypes (γδT cells, CD56 + T cells) compared with peripheral blood. A subset of DC-SIGN + macrophages, with a distribution adjacent to areas of placental attachment in contrast to the widespread setting of general CD68 + cells, was identified in both species. Together, these results demonstrate that the maternal–fetal interface in both cynomolgus and vervet monkeys is very rich in immune cells that have similar phenotypes to those seen in humans, indicating that both species are excellent models to study the contributions of distinct immune cell populations to pregnancy support.

The effects of tumor necrosis factor-alpha on in vitro differentiation of natural killer cells (138.13)

Abstract Natural killer (NK) cells are differentiated from hematopoietic stem cells (HSCs) in bone marrow. The differentiation of NK cells is regulated by various factors including soluble growth factors and transcription factors. Here, we demonstrated that tumor necrosis factor-α (TNF-α) is a positive regulator of NK cell differentiation. HSC-derived precursor NK (pNK) cells were further differentiated into mature NK (mNK) cells in the presence of IL-15 in vitro. The potential role of TNF-α on NK cell maturation was evaluated in the presence or absence of IL-15. TNF-α itself induced the expression of NK1.1 and CD122 of mature NK cells and its effects were significantly augmented in the presence of IL-15. IFN-γ production was maximal when NK cells were matured in the combination of IL-15 and TNF-α. Moreover mRNA expression of several transcription factors including T-bet and GATA-3 was increased during TNF-α -mediated in vitro NK cell differentiation. Overall, these data indicate that TNF-α regulates NK differentiation by increasing transcription factors which are crucial for NK maturation.

Regulatory role of NKp44, NKp46, DNAM-1 and NKG2D receptors in the interaction between NK cells and trophoblast cells. Evidence for divergent functional profiles of decidual versus peripheral NK cells

During the first trimester of pregnancy NK cells represent >50% of the lymphoid cells present in the human decidua where they reside in close contact with trophoblast cells. Because in decidual tissues NK cell activation and function may be induced by this interaction, we analyzed the cellular ligands recognized by activating NK receptors expressed on trophoblast cells. We show that these cells primarily express the NKp44 and DNAM-1 ligands and that interaction between these ligands and their corresponding receptors results in NK cell triggering. While activated peripheral blood NK (pNK) cells lysed the trophoblast cell lines JAR and JEG3, decidual NK (dNK) cells did not. On the other hand, they released VEGF, SDF-1, IP10 and large amounts of IL-8. Interaction with K562 target cells was exploited to induce optimal NK cell triggering, allowing a parallel, quantitative assessment of both cytolytic activity and cytokine production elicited by dNK cells. While dNK cells were unable to kill K562 even at high effector:target (E:T) ratios, they released large amounts of IL-8 also at low E:T ratios, a scenario compatible with dNK trophoblast cells interaction occurring within decidual tissues.

Phenotypic Differences Between Natural Killer Cells in Human Deciduas and Uterine Blood.

Background: Decidual natural killer (dNK) cells constitute the predominant leukocyte population at the time of human implantation, and modulate immune responses at the maternal-fetal interface in early pregnancy. The origin of dNK cells is unknown and they are proposed to arise from either proliferation of resident dNK cells or from an influx of peripheral blood NK (pNK) cells. The surface phenotype of dNK cells is similar to the minor CD56bright population of pNK cells, while these populations differ in expression of cytotoxic molecules. The phenotypic differences and cytotoxic armaments between NK cells from deciduas and uterine blood, however, remain unknown. Objectives: To characterize the surface phenotypes and cytotoxic granule expressions of NK cells from maternal deciduas and uterine blood during early gestation. Methods: Maternal decidual tissues and uterine blood were obtained from the first trimester termination of normal pregnancies (6-12 wk, n = 6-8) by suction curettage and used immediately for the isolation of the mononuclear cells (MCs) and NK cells by density gradient and Magnetic CD56+ sorting. Phenotypic and cytotoxic molecule characterization of the cells was performed by surface and intracellular flow cytometric analysis. Results: a) CD56+ NK cells were present in large numbers in the deciduas, and their percentage was significantly elevated when compared with that in uterine blood; b) the cells expressing CD94+, NKG2C+, and NKG2A+ increased significantly among MCs from deciduas compared with uterine blood; c) CD3+, CD45RO+, CD16+, and NKG2D+ cells were present at both deciduas and uterine blood, but the expression of these markers did not differ significantly between the two populations; and d) both NK cells in deciduas and uterine blood express substantial, but similar, amounts of the cytotoxic proteins perforin and granzyme B, but when compared to uterine blood NK cells, dNK cells express significantly greater quantities of the antimicrobial protein granulysin. Conclusion: Dramatic differences in phenotype exist between NK cells in deciduas and uterine blood in early pregnancy. The number of NK cells with NKG2A+ , a inhibitory receptor on cell cytotoxic effector function, are significantly increased in maternal deciduas, thus these cells may play a role in protecting the fetus from dNK cytolysis in a successful pregnancy. The mechanisms by which NK cells maintain their phenotypic differences between deciduas and uterine blood and the changes in their characteristics in recurrent pregnancy loss warrant further investigation.

CCL3 and CXCL12 regulate trafficking of mouse bone marrow NK cell subsets

AbstractHerein we have analyzed chemokine involvement in the trafficking of developing and mature mouse natural killer (NK) cells in the bone marrow (BM). We observed drastic changes of CCR1, CXCR3, and CXCR4 expression and function during progression from precursor NK (pNK) cells to immature DX5− NK (iNK) and mature DX5+ NK (mNK) cells. pNK and mNK cells expressed the 3 receptors, while only CXCR4 was detected on iNK cells. Correspondingly, mNK cells migrated to CXCL12, CXCL10, and CCL3, and pNK and iNK cells to CXCL12, whereas pNK cells migrated to CCL3 and CXCL10 only after CXCL12 stimulation. Comparison of BM, peripheral blood, and spleen mNK cell populations revealed that CXCL12, CXCL10, and CCL3 preferentially affected BM mNK cell migration. Administration of the CXCR4 antagonist, AMD-3100, to C57BL/6 mice induced strong reduction of mNK and iNK cells in the BM and increased their number in blood and spleen. Conversely, CCL3 administration selectively mobilized mNK cells from the BM and this effect correlated with its ability to inhibit CXCL12-mediated mNK cell responses in vitro. Our results suggest that the combined action of chemokines selectively regulates localization of NK cell subsets in the BM and direct their maturation and migration to the periphery.

Stage-dependent gene expression profiles during natural killer cell development

Natural killer (NK) cells develop from hematopoietic stem cells (HSCs) in the bone marrow. To understand the molecular regulation of NK cell development, serial analysis of gene expression (SAGE) was applied to HSCs, NK precursor (pNK) cells, and mature NK cells (mNK) cultured without or with OP9 stromal cells. From 170,464 total individual tags from four SAGE libraries, 35,385 unique genes were identified. A set of genes was expressed in a stage-specific manner: 15 genes in HSCs, 30 genes in pNK cells, and 27 genes in mNK cells. Among them, lipoprotein lipase induced NK cell maturation and cytotoxic activity. Identification of genome-wide profiles of gene expression in different stages of NK cell development affords us a fundamental basis for defining the molecular network during NK cell development.

Maternal-iron-deficiency effects on peritoneal macrophage and peritoneal natural-killer-cell cytotoxicity in rat pups

Cytotoxicity of peritoneal macrophages (pMs) and peritoneal natural killer (pNK) cells toward xenogenic tumor cells was studied in anemic, suckling rats. Dams were fed 6, 12, or 250 mg Fe/kg diet ad libitum throughout gestation and lactation. Pups were injected intraperitoneally with 10(5) plaque-forming units of virus. Four days later cytotoxicity of pMs and pNK cells against YAC-1 mouse lymphoma cells was measured. Body weight, hemoglobin, hematocrit, and viable cell yield of pups were significantly decreased with decreasing dietary iron. pM cytotoxicity was significantly impaired in anemic pups at pM-target-cell ratios of 10:1 and 30:1 at 4 and 16 h (P less than or equal to 0.03). pNK-cell cytotoxicity was significantly impaired in anemic pups at pNK-target-cell ratios of 10:1 and 50:1 at 16 h. Iron-deficient diet consumed by dams throughout gestation and lactation resulted in anemic offspring whose immunologic defense by pMs and pNK cells against xenogenic tumor cells was significantly reduced.